Oxidative stress protein Oxr1 promotes V‐ATPase holoenzyme disassembly in catalytic activity‐independent manner

The vacuolar ATPase (V‐ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V(1)‐ATPase and V(o) proton channel subcomplexes. Here, we report cryo‐EM structures of yeast V‐ATPase assembled in vitro from lipid nanodisc reconstituted V(o) and mutant V(1). Our analysis identified holoenzymes in three active rotary states, indicating that binding of V(1) to V(o) provides sufficient free energy to overcome V(o) autoinhibition. Moreover, the structures suggest that the unequal spacing of V(o)’s proton‐carrying glutamic acid residues serves to alleviate the symmetry mismatch between V(1) and V(o) motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V(1) bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V(1)‐ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V‐ATPase regulation.